Epileptic disorders affect about 20 to 40 million people worldwide. Generalized idiopathic epilepsies (IGE) cause 40% of all epileptic disorders and commonly have a genetic basis (Plouin, 1994). Most of the IGEs that are inherited are complex, non-monogenic diseases. One type of IGE is Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited disorder of newborns (Ronen et al., 1993; Hauser and Kurland, 1975). BFNC (OMIM 121200) is an autosomal dominantly inherited epilepsy of the newborn infant. This idiopathic, generalized epilepsy typically has an onset of seizures on day two to four of life. Spontaneous remission of the seizures occurs between two to fifteen weeks (Ronen et al., 1993; Plouin, 1994; Hauser and Kurland, 1975). Seizures typically start with a tonic posture, ocular symptoms and other autonomic features which then often progress to clonic movements and motor automatisms. These neonates thrive normally between the seizures, and their neurologic examinations and later development indicate normal brain functioning (Ronen et al., 1993; Plouin, 1994; Hauser and Kurland, 1975). However, in spite of normal neurologic development, seizures recur later in life in approximately 16% of BFNC cases compared with a 2% cumulative lifetime risk of epilepsy in the general population (Ronen et al., 1993; Plouin, 1994; Hauser and Kurland, 1975).
Genetic heterogeneity of BFNC has been observed (Ryan et al., 1991). Two loci, EBN1 and EBN2, have been mapped by linkage analysis to chromosome 20q13 (Leppert et al., 1989; Malafosse et al., 1992) and chromosome 8q24 (Lewis et al., 1993; Steinlein et al., 1995), respectively.
The nomenclature of the genes of this invention as well as related genes has changed over time. Two of the genes of this invention from humans are now referred to as KCNQ2 and KCNQ3. These had originally been named KVEBN1 and KVEBN2, respectively. The two sets of names are equivalent and can be used interchangeably, but the accepted nomenclature is now KCNQ2 and KCNQ3 and these names will be used herein. Also, the related gene KCNQ1 had originally been called KVLQT1 in the literature, but again the accepted name now is KCNQ1 and this name will be used herein.
Linkage analysis in a large kindred demonstrated that a gene, herein called KCNQ2, responsible for BFNC maps to chromosome 20q13.3 close to the markers D20S20 and D20S19 (Leppert et al., 1989). Following the initial report, two centers confirmed linkage of BFNC to the same two genetic markers on chromosome 20, termed the EBN1 (epilepsy benign neonatal type 1) locus (Ryan et al., 1991; Malafosse et al., 1992; Steinlein et al., 1992). A more distal marker, D20S24, shows complete co-segregation with the BFNC phenotype in chromosome 20 linked families. Finding a distal flanking marker for the BFNC locus has not been successful probably because of its proximity to the telomere. This telomeric region is characterized by a high recombination rate between markers when compared to the physical distance (Steinlein et al., 1992). In fact, Steinlein et al. have demonstrated that the three markers D20S 19, D20S20 and D20S24 are contained on the same 450 Mb Mlu I restriction fragment (Steinlein et al., 1992). All of the families in the present study used to find and study KCNQ2 show linkage to chromosome 20q markers with LOD scores of greater than 3.0 or have probands with clinical manifestations consistent with BFNC (Leppert et al., 1993). Each subject and control signed a Consent for Participation in these studies approved by the Institutional Review Board for Human Subject Research at their home institution. To find a gene responsible for BFNC, we narrowed a BFNC region with a sub-microscopic deletion in a single family, identified candidate cDNAs in this deletion, and then searched for mutations in other BFNC families. The gene has been identified and sequenced. Several distinct mutations have been found in this gene. These include a large deletion, three missense mutations, three frameshift mutations, two nonsense mutations and one splice site mutation. One of these mutations is associated with rolandic epilepsy as described in the Examples below.
A second chromosomal locus, EBN2, has also been identified for BFNC. Lewis et al. (1993) demonstrated linkage to markers on chromosome 8q24 in a single Hispanic family affected with BFNC. Evidence for this second locus was also reported in a Caucasian pedigree (Steinlein et al., 1995). The gene, herein called KCNQ3, responsible for EBN2 was mapped to chromosome 8, between markers D8S256 and D8S284 on a radiation hybrid map (Lewis et al., 1995). KCNQ3 has been identified as set out in the examples of the instant disclosure. KCNQ3 was screened for mutations in the large BFNC family previously linked to chromosome 8q24 in the same marker interval (Ryan et al., 1991; Lewis et al., 1993). A missense mutation was found in the critical pore region in perfect cosegregation with the BFNC phenotype. The same conserved amino acid is also mutated in KCNQ1 in an LQT patient (Wang et al., 1996). Furthermore, the segment of mouse chromosome 15 that harbors the stargazer (stg) locus (Noebels et al., 1990; Letts et al., 1997) is homologous to the human 8q24 region and the stg phenotype is close to a common form of IGE, the absence epilepsy. KCNQ2, KCNQ3 and other undiscovered genes of the same family of K+ channels are strong candidates for other, more common IGEs. One individual with juvenile myoclonic epilepsy has been found who has a mutation in an alternative exon of KCNQ3 as shown in the Examples below.
IGEs include many different types of seizures. Common IGEs include generalized tonic-clonic seizure (GTCS), absence epilepsy of childhood (AEC), juvenile absence epilepsy (JAE) and juvenile myoclonic epilepsy (JME). Reutens and Berkovic (1995) have shown that the boundaries between the different IGE syndromes are indistinct and suggest that neurobiological and possibly genetic relationships exist between these syndromes. Interestingly, using non-parametric linkage methods, Zara et al. (1995) obtained evidence for involvement of an epilepsy locus at chromosome 8q24 in a panel of families with multiple cases of IGEs. Furthermore, in a population study, Steinlein et al. (1997) recently described a weak allelic association at the CHRNA4 locus, on chromosome 20q13.3, physically close to KCNQ2, in a group of unrelated patients with multiple forms of IGEs. Finally, an epileptic mutant mouse stargazer (stg) (Noebels et al., 1990) is a genetic model of spike wave epilepsy. This is a recessive mutation and the phenotype is related to a common form of human IGE, the absence epilepsy. Stg has been mapped on mouse chromosome in a region homologous to the human 8q24 region. Screening the mouse homolog of KCNQ3 for mutations in an affected mouse will assess the hypothesis that the same gene is responsible for both BFNC and Stargazer phenotypes.
The present invention is directed to both KCNQ2 and KCNQ3 and their gene products, mutations in the genes, the mutated genes, probes for the wild-type and mutated genes, and to a process for the diagnosis and prevention of BFNC. Each of the genes encodes a potassium channel protein. The instant work shows that some families with BFNC have mutations in either KCNQ2 or KCNQ3. BFNC is diagnosed in accordance with the present invention by analyzing the DNA sequence of the KCNQ2 and/or KCNQ3 gene of an individual to be tested and comparing the respective DNA sequence to the known DNA sequence of a normal KCNQ2 and/or KCNQ3 gene.
Alternatively, the KCNQ2 gene and/or KCNQ3 gene of an individual to be tested can be screened for mutations which cause BFNC. Prediction of BFNC will enable practitioners to prevent this disorder using existing medical therapy. Furthermore, a mutation in KCNQ2 has been found which is associated with rolandic epilepsy and a mutation in KCNQ3 has been found which is associated with JME. These two forms of epilepsy may also be diagnosed in accord with the invention.
Mouse genes homologous to the human KCNQ2 and KCNQ3 have also been found and sequenced and the sequences are disclosed. The mouse KCNQ2 gene has been only partially isolated and sequenced (shown as SEQ ID NO:88), the 3xe2x80x2 end not yet having been found. The complete mouse KCNQ3 gene has been isolated and sequenced (shown as SEQ ID NO:90).
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
The present invention demonstrates a molecular basis of Benign Familial Neonatal Convulsions (BFNC) as well as for rolandic epilepsy and juvenile myoclonic epilepsy. More specifically, the present invention has determined that molecular variants of either the KCNQ2 gene or KCNQ3 gene cause or are involved in the pathogenesis of these three forms of epilepsy. Genotypic analyses show that KCNQ2 is linked to BFNC in ten unrelated families and KCNQ3 is linked to BFNC in one other family. Furthermore, one mutation in the KCNQ2 gene in two individuals of one family has been associated with rolandic epilepsy and one individual with a mutation in KCNQ3 has been diagnosed with juvenile myoclonic epilepsy. Analysis of the KCNQ2 and KCNQ3 genes will provide an early diagnosis of subjects with BFNC, rolandic epilepsy or JME. The diagnostic method comprises analyzing the DNA sequence of the KCNQ2 and/or the KCNQ3 gene of an individual to be tested and comparing it with the DNA sequence of the native, non-variant gene. In a second embodiment, the KCNQ2 and/or KCNQ3 gene of an individual to be tested is screened for mutations which cause BFNC, rolandic epilepsy or JME. The ability to predict these epilepsies will enable physicians to prevent the disease with medical therapy such as drugs which directly or indirectly modulate K+ ion channels.
The invention shows that various genetic defects of a potassium channel are responsible for the human idiopathic epilepsy of BFNC, rolandic epilepsy and/or JME. This finding adds to the growing list of channelopathies in humans (Ptacek, 1997). Importantly, this result suggests that drugs which directly or indirectly modulate K+ ion channels will be helpful in the treatment of seizure disorders.